This proposal continues studies that use combined physiological and ultrastructural analysis to develop a detailed understanding of vasopressin action in the toad urinary bladder. The following new strategies developed in the last grant period will be used to elucidate the role of membrane structural features in the water permeability response: 1) "Sandwich" replica analysis combines features of freeze-fracture and deep-etching to provide a unique view of the interrelationship between organized elements on the surface of membranes and intramembrane structure 2) "Hyperstretch" of bladders incubated in ice-cold Ringer flattens the apical membrane nearly completely to allow a comprehensive evaluation of the incidence and area of intramembrane particle aggregates in vasopressin-treated bladders 3) "Label-fracture" analysis of extracellular markers endocytosed with removal of vasopressin demonstrates that intact aggregates are retrieved from the apical surface with conditions that reduce the apical membrane permeability response. Novel applications of this method now allow two types of aggregate containing membrane vesicles to be distinguished in the cytoplasm following vasopressin removal and permit a quantitative evaluation of the role of membrane retrieval in regulation of the permeability response. In addition, methods that allow selective labeling of retrieved membrane constituents have led to a promising new strategy for isolation of these structures and the identification of aggrephore-specific proteins. Using antibodies to apical membrane proteins we will evaluate the specificity of membrane retrieval responses. We will use monoclonal antibodies to aggrephore components and label-fracture methods to localize these components with respect to the intramembrane particle aggregates. This strategy should allow us to distinguish which protein(s) of the complex aggrephore vesicles directly relates to the aggregates and therefore to the channels of interest. In addition, labeling studies are described to evaluate the extent to which aggrephores recycle during continuous vasopressin stimulation or in a subsequent response following removal of hormone.